Production of organic acids



Patented Apr. 6, 1954 2,674,551 ICE PRODUCTION OF ORGANIC ACIDS Andrew J. Meyer, Peoria, 111., assignor to the United States of America as represented by the Secretary of Agriculture No Drawing. Application September 8, 1950, Serial No. 183,890

(Granted under Title 35, U. S. Code (1952),

see. 266) 22 Claims.

The invention herein described may be manufactured and used by or for the Government of the United States of America for governmental purposes throughout the. world without the pay- ;ment to me of any royalty thereon.

fermentable carbon sources, assimilable nitrogen sources, together with the various known necessary inorganic nutrients and trace elements. The prior art production of these acids bymold "fermentation results in disadvantageously low yields, considerably less than the theoretical based upon the carbonv source metabolized by the molds. This disadvantage can be accounted for in part by the formation of other organic acids which not only reduce the yield of the desired acids but often impair the process of recovery and separation.

An object of this invention is to provide a process whereby'the desired acids are produced inincreased yields, and the production of undesired products is substantially inhibited. A further object is to provide a process whereby difficulties in the recovery of the desired acids, encountered by prior workers, are reduced or eliminated and whereby the acids can be produced more economically by the use of more impure nutrient sources.

ihe objects of this invention are accomplished by the use of methyl acetate or low molecular weight monohydric alkanols as adjuncts to the fermentation media. The effect of these alkanol effective as other alkanols; nevertheless, produc ing marked increases.

According to my invention, methyl acetate. methanol, ethanol, and the propanols are added to the ferementation media in amounts ranging from 1 to 5 percent by volume, preferably 1 to I percent. The fermentations are otherwise carried out in general in accordance with known methods. For example, the same general type of 1 nutrient media is employed, containing the usual fermentable carbon sources, nutrients, etc., with the exceptions which will be described hereinafter. The same organisms are employed as are known in the art to produce the desired acids. The fermentations may be carried out either by surface or by submerged methods.

The effect of the adjuncts in producing superior yields of acid is not general for the whole class of acid-producing fermentations. Insofar as I have discovered it is not possible to predict the effects on the acid production, on the vegetative growth of the mold or on the stimulation or inhibition of undesired metabolic products. For example, neither methanol, ethanol or propanol will increase the yield of kojic acid to any material extent. The yield of gluconic acid is actually reduced by the alkanol adjuncts.

I have discovered during the course of my investigations that the relation between the vegetative growth of the organism and optimum'acid production, according to my process, is contrary to what would normallybe expected. In general, the effect of adjuncts is to decrease the mycelium growth when compared with a control fermentation. In a few isolated cases I have noted a slight temporary stimulation of mycelium growth, but this has always disappeared as the fermentation continued, the net result being a decrease. The physiological efi'ect of the adjunct appears to be a toxicant, reducing both vegetation and tendency toward sporulation. If employed in amounts in excess of my ranges, the effect is to reduce materially both growth of the organism and acid production. However, within the ranges specifled, even though attended by a small decrease in mycelium growth, the desired acids are produced in surprisingly increased yields, and the production of undesired metabolic byproducts materially reduced.

The advantages attending my invention are not'limited to an increase in yield of the acids, but comprise other valuable features. For example, when employing my invention, the fermentation may be carried out successfully without the necessity for using pure nutrients or for elimination of trace elements, such as manganese in the case of citric acid. Furthermore, the fermentations are not so sensitiv to pH conditions. as in prior methods. According to my process the fermentations may be carried out within the initial pH ranges of 1.5 to 8.5. This latter feature is of particular value in using molasses as the carbon source in citric acid production, since it is difiicult to lower the pH to the range of optimum condition as heretofore known.

Citric acid may be produced according to my invention employing any known citric acidproducing organism. Examples are Aspergillus niger, A. wentii, A. schiemanm', Penicillium implicatum. and the like. The carbon sources may be glucose, sucrose, molasses, such as beet molasses and blackstrap molasses, polyhydrlc alcohols, starch or modified starches, ground grain, and the like. In the case of itaconic acid the organism employed may be varieties of Aspergillus terreus with nutrients similar to those employed in the case of citric acid. Ethylene oxide dicarboxylic acid is produced by such organisms as Aspergillus fumigatus. The carbon source may be commercial glucose or other carbohydrate material known to produce this particular acid.

The following examples are illustrative of my invention.

EXAMPLE 1 This example illustrates the effect of methanol, ethanol, and isopropanol on the production of ethylene oxide dicarboxylic acid, recovered as the calcium salt.

Fifty ml. portions of a fermentation medium 4 were prepared in a series of 200 ml. Erlenmeyer flasks. The medium consisted of the following ingredients made up for one liter:

Grams Glucose monohydrate 150.0 Beet molasses 20.0 Corn steep liquor 10.0 MgSOr-lHzO 0.25 (NH4)2SO4 0.70 KH2PO4 0.30 ZnSO4-7H2O 0.044

To each culture was added 1.5 g. of CaCOa. Just prior to inoculation adjuncts of the various alcohols were made to the sterile media. Inoculation was accomplished with dry, ungerminated spores of Aspergillus fumlgatus. The flasks were in- ;cubated for 12 days at 30 C. The results are summarized in the following table.

Table I Calcium salt of ethylene oxide dicarboxylic acid per culture Alkanol adjuncts, by volume Grams EXAMPLE 2 This example illustrates the effect of methanol on the production of ethylene oxide dicarboxylic acid in a series of experiments employing various carbon sources. The fermentation organism was Aspergzllus fumigatus.

Fifty ml. portions of a fermentation medium were prepared in a series of 200 m1. Erlenmeyer flasks. The medium consisted of the following ingredients made up for one liter:

Carbon source-as designated in Table II.

Grams Beet molasses 20.0 Corn steep liquor 15.0 MgSO4-7H2O 0.250 KHzPOr 0.300 ZnSOy'II-IzO 0.088 (NI-I4) 2804 0.700

The flasks were inoculated with Aspergillus fumigatus. Immediately before inoculation the adjunct was added to half of the flasks. The flasks were incubated for days at 30 C. The results are summarized in Table II.

This example illustrates the effect of ethyl alcohol on the production of itaconic acid by Aspergillus terreus. Fifty ml. portions of a fermentation medium were prepared in a series of 200 ml. Erlenmeyer flasks. The following base medium was employed:

Glucos monohydrate g 110.0 KHzPOs g 0.100 MgSO4-7HzO g 0.500 NH4NO3 g 1.50 ZnSOi-7HzO g 0.044 Argo corn steep liquor ml 3.0

The fermentation media had an initial pH of 4.1. The flasks were inoculated with Aspergillus terreus. Immediately before inoculation, the adjuncts were added as designated in the table below. The flasks were incubated for 7 days. The results are summarized in Table III.

Table III l/l0 Gl Tit,ield 9 Th i ucose l acomc eory z g g itaconic 32; of consumed acid yield based percent acid per m cenum per based on on glucose culture y culture glucose consumed consumed Weight Ml. Grams Grams Percent Percent *1 mole glucose 1 mole itoconic acid.

EXAMPLE 4 This example illustrates the efiect of methanol and isopropano1 on the production of itaconic acid. Fifty ml. portions of the fermentation medium were prepared as in the above examples. The medium consisted of the following ingredients made up for one liter:

Sucrose g 121.0 MgSOr'THzO g 0.250 KHzPOe g 0.100 NH4NO3 g 1.00 Mg(NO3)2-4H2O g 1.00 ZnSO4'7H2O g 0.044 Corn steep liquor ml 2.0

The initial pH of the medium was 4.0. The flasks were inoculated with ungerminated spores of Aspergillus terreus. Immediately before inoculation, the adjuncts were added as designated in the table below. The flasks were incubated for 9 days, The results are summarized in Table IV.

Table IV EXAMPLE 7 Finelyground com (140 g.) was employed in ggg Dry a medium comprising nutrient salts and 5.0 Adjunct by Wlume acid per 5 grams of CaCOs. One hundred ml. portions were culture employed in 300 ml. Erlenmeyer flasks. After steam sterilizationthe flasks were inoculated with None 8 f 25 3 ml. of a suspension of germinated spores. To g? 340 0.77 half of the cultures was added 4 percent methg a lo anol as the adjunct. The flasks were incubated for 9 days on a Ross-Kirshaw shaker. The re- EXAMPLE 5 sults are summarized in Table VII.

This example illustrates the elfect of methanol 0n the production of itaconic acid in submerged Table VII cultures. One hundred m1. portions of the fermentation medium were prepared in 300 m1. N/m citric oxalic Gluconic flasks. The medium consisted of the following Methanol, volume percent acid p acid acid ingredients made up for one liter: culture present present Glucose monohydrate g 165.0 m KHZPOQ g 0.1.00 0 181 None Trace. MgSO4-7H2O g 0.500 4 3 NH4NO3 g 1.50

ZnSO4-7H2O g 0.044 Argo corn steep liquor ml 3.0

The initial pH of the medium was 4.1. The EXAMPLE 3 i flasks were inoculated with ungerminated spores Elevep gram Pf of 3 corn starch were of Aspergillus terreus. Immediately before inocplaced m a Senes 300 El'lenmeyerfiasksulation, the adjunct was added to one-half of To {each flask W added 90 m1 of a nutrient the cultures. A submerged f m nt ti n was medium containing the following ingredients: carried out for a period of 9 days. The results are summarized in Table V. $3 7H2O g 0.03 Table V NHiNOs g 0.075 IhPO4(0.5 N) .ml 3.0 /m. Dry weight HzSO4(0.5 N) mi 2.0 Methanol, volu e perce t 553%; Corn steep liquor 'ml 0.7 culture mycehum ZnSO -7H Q g 0,004 Glucose monohydrate g 0.1 Ml. Grams g 40 The flasks were heated in a water bath with stirring until the starch had gelatinized. The flasks were then sterilized in an autoclave at EXAMPLE 6 20 pounds steam pressure for 30 minutes. After This example illustrates the efiect of methanol sterilization t medium had an initial pH of as n adju in the production of Citric a 3.0 and contained 0.21 percent glucose. To defrom sucrose by various Aspergilli in surface cul- 4 5 termine t potential glucose of th i a t Fifty ml. portions of the fe t n sample was given a strong acid hydrolysis wheremediu W P ep d in Examples 1 t9 upon it analyzed 10.84 percent glucose. Each The medium consisted Of the following ingredlflask was eeded at room temperaturewithla n made p for One liter: small portion of a germinated spore inoculant Sucrose g 164.0 59 of Aspergillus niger. Sufiicient sterile water was MgSO4-7H2O g 0.50 added to make a volume of 100 ml. per culture for KH POJ g 0.15 half the cultures. To the remaining half of the Nog g 1.50 cultures was added 3 volume percent methanol 21 30 0 g 0.044 r based on ml. volumes. These cultures were Corn steep liquor m1 6-0 then made up to 100 ml. with sterile water.

The initial H of the medium was 4.2. The The fi k w re incubated f r 8 y t 30 flasks were inoculated with various Aspergilli im- C 011 a ROSS-KiI'ShaW Shaker- The results are mediately after addition of the adjunct tooneta u ted in Table VIII- half of the cultures, and were incubated at 30 C. CO gilr 8 days. The results are summarized in Table Table VIII Table VI Glucose remaining per Nllon culture f (,5 Methacitric N/l 0 citric citric acld A. mger strain n 0} acid per Mold used Methanol g ggg culture Before fg ggg on u consumed hydrolysls hydrolysis Volume Weight Volume percent Ml. percent '(6 Percent Ml, Grams Grams me 8 iii i333 N-RRL 330 2 332 3:83 31?? A. were 3 5% i253 NRRL 3 433 3351i 3333 A. schz'emrm'ni g N'RRL g 31 EXAMPLE 9 Sufficient hydrochloric acid was added to 200 g. of beet molasses to give a pI-I of 7.2. The molasses had been treated with potassium ferrocyanide to remove such trace elements as iron and manganese and had an initial sucrose content of 54 percent. The acidified molasses was made up to one liter with water, and 50 ml. portions were placed in 200 ml. Erlenmeyer flasks and steam sterilized. When cool, varying amounts of methanol were added as adjuncts. After which, the flasks were inoculated with ungermlnated spores of Aspergillus niger. The flasks were incubated for 8 days at 30 C. The results are summarized in Table IX.

EXANEPIE 10 The following base medium was employed, the basis being one liter:

Glucose g 126.0 NH4NO3 g 1.75 K2804 g 0.232 NH4H2P04 g 0.400 ZnSO4-7HzO g 0.044 Ferric tartrate g 0.005 Corn steep liquor ml 0.5

Fifty ml. portions of this medium were placed in 200 ml. Erlenmeyer flasks. The adjuncts, as designated in Table X, were added to the base medium just before inoculation with ungerminated spores of Aspergillus niger. All cultures were incubated at 30 C. for 6 days. The results are summarized in Table X.

Table X YLielid of an y rous Adigllncts to thclbase y g ggg g 2 ,2515 cgtricdacid me rum, 1} v umc ase on culture pe. culture glucose consumed Ml. Grams Weight percent 54 .25 7. 4

h 8 EXAMPLE 11 Two media consisting of the following ingredients were prepared:

Grams Glucose 11.34. MgClz- 6H2O 0.020 IIaH2PO4 0.020 KCl 0.010 MQSO47H2O 0.005 ZnSOr '7TI-I2O 0.004 Ferric tartrate 0.001 MnSO4-4I-I2O 0.005 Corn steep liquor 0.020 NaNoa 0.162 NH4C1 0.013

Distilled water to make m1.

To one medium was added 3 ml. of methanol. Both media were then inoculated with 2 ml. of a germinated spore suspension of Aspergillus niger. The media were fermented on a Ross- Kirshaw shaker at 30 C. for 10 days. The results are summarized in Table XI.

This example illustrates the effect of methanol adjunct in varyin amounts upon the production of citric acid from commercial glucose (glucose monohydrate) by Aspergzllus niger. The initial pH of the media in this example was obtained by the addition of 0.482 N hydrochloric acid. The following base medium was employed, the basis being one liter:

Glucose monohydrate g 160.0 NH4NO3 g 1.750 K2SO4 g 0.232 NH4H2PO4 g 0.400 MgSO4-7H2O .g 0.250 ZnSO4-7H2O g 0.044 Ferric tartrate g 0.005 Corn steep liquor ml 0.5

Six cultures of 50 ml. each of this medium were placed in 200 ml. Erlenmeyer flasks. Hydrochloric acid was added to 5 in amounts sufiicient to obtain the hydrogen ion concentrations indicated in Table XII (a). After fermentation, the following results were obtained.

Table XII (a) N/lO citric Dry weight Yield Initial pH value ga g acid per of myceliurn fi g culture per culture cgnsumed Ml. Grams The initial procedure of this example was reheated. withhowever. the addition of one per- 9 cent methanol by volume to the media just prior to inoculation. The results are summarized in Table XII (1)).

Table XII (b) Yield 1\'/lO citric Dry weight Initial pH value :65? acid per of mycelium gh g g culture per culture consumed Weight Ml. Grams percent 0 258 1.18 31.2 10 s 1. 2s .20 350 1. 24 40.11 392 1. 24 44.1 '424 1.27 1 45.1 1 50 532 1.14] 63.2 lb

The initial procedure of this example was repeated, except that 2 percent methanol by volume was added to the media just prior to inoci ulation. The results are summarized in Table 50 XII (c).

' Table XII (c) Yield N/lO eltrrc Dry weight 9 Initial pH value sag i acid per 01 mycclium fifgg culture per culture cfnsumed Weight Ml. Grams percent 0 476 1.12 47.5 30 10 510 1.13 54.1 20 542 1.08 57.5 30 508 1.09 55.2 40 496 1.02 55.5 50 494 0. 96 56.8

'5 The initial procedure of this example was repeated, except that 3 percent methanol by'volume was added to the media just prior to inoculation. The results are summarized in Table XII (d). 40

Table XII (d) Yield N/lO cltrlc Dry weight Initial pH value 056331 acid per ofrnycelium fig gg culture per culture consumed Weight Ml. Grams percent 0 518 1.03 50.3 10 454 0.87 59.9 20 444 0.87 00.0 so 434 0. 80 59.8 40 412 0.72 60.2 50 312 0. 62 54.8

As previously mentioned, my invention makes possible the use of cruder raw materials in the fermentation media than has heretofore been possible for the satisfactory production of the acids. The high yields of the desired products, characterizing my invention, are not diminished by the presence of trace elements heretofore thought to be deleterious. This is particularly true of the production of citric acid from crude molasses. The molasses vary greatly in trace element content; for example, the manganese content may vary from 5 to parts per million, depending on the source andprevicus treatment. This element is one well known to interfere in prior fermentation processes for citric acid production. In my process the necessity for purification and removal of manganese from crude molasses is obviated.

' Example 11 above illustrates a tolerance for appreciable amounts of manganese. The following examples illustrate the invention employing controlled amounts of manganese, and it .10 may be seen that relatively large amounts of this element can be tolerated without diminution in citric acid production.

Example 13 illustrates the production of citric acid from a sucrose medium containing high concentrationsof manganese, supplied as manganese sulfate, employing 3 percent methanol as the adjunct. The effect of all 5 adjuncts over the ranges of 1 to 5 volume percent is similar to the results of these examples. Example 14- illustrates the production of citric acid from beet molasses. The crude molasses were treated with potassium ferrocyanide to remove such trace elements as manganese and. iron. Manganese sulfate was then added to the media in the designated amounts.

EXAMPLE 13 Fifty ml. portions of a fermentation medium were prepared. The medium consisted of the following ingredients made up for one liter:

Sucrose I g 160.0 NH4NO3 g 1.50 KH2PO4 g 0.150 Corn steep liquor ml 3.0 ZIISO47H2O g 0.044

The initial pH of the medium was 4.1. Each flask was sterilized and then inoculated with spores of Aspergillus niger and then incubated at 30 C. for 8 days. The results are summerized in Table XIII.

Table XIII D h lE'igld of anry weg t y rous cit- Manglaitiese 3 5 lof myce- {Die based p-r 1 er rum per ase on n culture culture sucrose consumed Weight Mg. Ml. Grams Percent EXAMPLE 14 Table XIV N /l[) citric Manganese Glucomc per liter Methanol 1335;: Spore acid present Volume Mg. Percent Ml.

0 O 402 None Fair 0 0 545 None Trace 10 0 209 Heavy Good 10 2 455 None Trace In this example the addition of manganese in the absence of methanol caused heavy sporulation and pronounced decrease'in acid production. Moreover, the addition of the adjunct increased the production of citric acid and reduced the amount of gluconic acid formed.

Examples 15 and 16 which follow illustrate my invention employing blackstrap molasses as the carbon source. Example 15 illustrates my process using submerged methods, while Example 16 illustrates a surface method.

EXAMPLE 15 This example illustrates the effect of methanol and ammonium chloride on the production of citric acid from blackstrap molasses by Aspergillus nicer in submerged culture.

The medium consisted of 288 grams of blackstrap molasses containing 150 grams of invert sugar and 0.044 gram of zinc sulfate per liter. The molasses had an initial pH of 5. 1. Each culture consisted of 100 ml. of this medium, inoculated with 2 ml. of a germinated spore suspension in 300 ml. Erlenmeyer flasks. incubated at 28 C. on a Gump shaking machine at 200 R. P. M. for 10 days. The methanol was added just before inoculation.

In a comparative experiment only traces of This example illustrates the efiect of methanol on the production of citric acid from blackstrap molasses by Aspergillus niger in surface culture. The cultures consisted of milliliters of nutrient solution in 200 ml. Erlenmeyer flasks inoculated with dry ungerminated spores. The only nutrient material was 288 grams of blackstrap molasses (150 grams of invert sugar) per liter. The germination was allowed to proceed for 8 days with the results tabulated below.

Table XVI Percent methanol added at start N [10 acid per culture, m1 176 460 620 706 698 Dry weight of mycelium per culture, g 1.10 1.06 1.14 1.09 1. 11 Weight yield of citric acid based on sugar consumed, percent 28 59 64 70 72 Spores on mycelium Heavy Trace Trace None None I claim:

1. A method for increasing the yield of an organic acid of the group consisting of citric acid. itaconic acid, and ethylene oxide dicarboxylic acid by mold fermentation of fermentable carbon sources comprising carrying out the fermentation in the presence of 1 to 5 percent by volume of one of the group consisting of low molecular weight monohydric alkanols and methyl acetate.

2. Process of claim 1 carried out under submerged-aerated conditions.

3. Process of claim 1 carried out by surface cultivation.

The flasks were 4. The method of claim 1 in which the fermentation medium contains corn steep liquor and is carried out under submerged-aerated conditions.

5. Method for the production of an organic acid of the group consisting of citric acid, itaconic acid, and ethylene oxide dicarboxylic acid by mold fermentation which comprises cultivating a mold known to produce the acid in a medium containing a fermentable carbon source and 1 to 5 volume percent of an adjunct of the group consisting of methanol, ethanol, normal propanol, isopropanol and methyl acetate.

6. A method for the production of citric acid by mold fermentation which comprises cultivating a citric acid producing mold in a medium containing a fermentable carbon source and 1 to 5 volume percent of an adjunct of the group consisting of methanol, ethanol, normal propanol, isopropanol and methyl acetate.

7. Method of claim 6 in which the fermentation is carried out within the range of pH 1.5 to pH 8.5 and the fermentable carbon source is glucose.

8. Method of claim 6 in which the fermentation is carried out within the range of pH 1.5 to pH 8.5 and the fermentable carbon source comprises sucrose.

9. Method of claim 6 in which the fermentation is carried out within the range of pH 1.5 to pH 8.5 and the fermentable carbon source is molasses.

10. Method of claim 6 in which the fermentation is carired out within the range of pH 1.5 to pH 8.5 and the fermentable carbon source is blackstrap molasses.

11. Method of claim 6 in which the fermentation is carried out within the range of pH 1.5 to pH 8.5 and the fermentable carbon source comprises starch.

12. Method of claim 6 in which the fermentation is carried out within the range of pH 1.5 to pH 8.5 and the fermentable carbon source is ground corn.

13. Process according to claim 6 carried out under submerged-aerated conditions.

14. Process of claim 6 carried out by surface cultivation.

15. The method of claim 6 in which the mold is taken from the group consisting of Aspergillus niger, Aspergillus wentz'i, Aspergzllus schz'emanm', and Penicillium implicatum.

16. Method for the production of itaconic acid which comprises cultivating Aspergillus terreus in a medium containing a fermentable carbon source and 1 to 5 volume percent of an adjunct of the group consisting of methanol, ethanol, notrmal propanol, isopropanol and methyl ace- 17. Method of claim 12 in which the fermentation is carried out within the range of pH 1.5 to pH 8.5, the fermentable carbon source is glucose and the adjunct is methanol.

18. Method for the production of ethylene oxide dicarboxylic acid which comprises cultivating Aspergz'llus fumigatus in a medium containing a fermentable carbon source and 1 to 5 volume percent of an adjunct of the group consisting of methanol, ethanol, normal propanol, isopropanol and methyl acetate.

19. Method of claim 17 in which the fermentation is carried out within the range of pH 1.5 to pH 8.5, the fermentable carbon source comprises glucose and the adjunct is an alkanol.

20. Method for the production of citric acid by mold fermentation which comprises cultivating a, citric acid producing mold in a medium References Cited in the file of this patent containing a fermentable carbon source, appre- UNITED STATES PATENTS ciable amounts of manganese, and 1 to 5 volume percent of an adjunct of the group consisting Numbel Name Date of low molecular weight monohydric alkanol and 5 2,006,036 May June 1935 methyl acetate. 2,416,821 Coulthard Mar. 4, 1947 21. The method of claim 20 in which the me- 2,438,136 $21168 1948 dium contains 5 to 100 parts per million of 2362981 Lockwood 1949 manganese.

22. The method of claim 20 in which the ad- 10 OTHER REFERENCES junct is methanol, Foster, Chemical Activities of Fungi, 1949,

Academic Press Inc., Pub., New York, New York, pages 400-7. 

1. A METHOD FOR INCREASING THE YIELD OF AN ORGANIC ACID OF THE GROUP CONSISTING OF CITRIC ACID, ITACONIC ACID, AND ETHYLENE OXIDE DICARBOXYLIC ACID BY MOLD FERMENTATION OF FERMENTABLE CARBON SOURCES COMPRISING CARRYING OUT OF THE FERMENTATION IN THE PRESENCE OF 1 TO 5 PERCENT BY VOLUME OF ONE OF THE GROUP CONSISTING OF LOW MOLECULAR WEIGHT MONOHYDRIC ALKANOLS AND METHYL ACETATE. 